Research Article www.acsami.org
On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging Samuel S. Hinman,† Charles J. Ruiz,‡ Georgia Drakakaki,§ Thomas E. Wilkop,*,§ and Quan Cheng*,†,‡ †
Environmental Toxicology and ‡Department of Chemistry, University of California, Riverside, Riverside, California 92521, United States § Department of Plant Sciences, University of California, Davis, Davis, California 95616, United States S Supporting Information *
ABSTRACT: The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, highthroughput manner. Various protein−ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents. KEYWORDS: SLB, HBM, SPR, SPR imaging, devitrification, microarray
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receptors.5,6 Thus far, SLB systems have been used for a variety of applications, including gaining insight into biophysical processes,7,8 enhancing drug delivery through incorporation of synthetic receptors,9 and designing sensors that bind molecules to their natural targets.10−12 Despite the potential and flexibility of SLBs, microarray applications of these systems have been scant. This is in large part due to the complexity and limited scalability of generating and maintaining SLBs in an aqueous environment in a way that ensures membrane integrity and unaltered activity of embedded components. Currently, common methods of creating patterned lipid bilayer microarrays include utilizing lipid corrals,13 utilizing noncontact printing through confined films,14 and injecting single-composition vesicle suspensions over multielement array substrates.15 Each of these methods
INTRODUCTION The cell membrane is a fundamental structure of living organisms, separating exterior and interior content, with embedded receptors and structures facilitating communication and regulated active and passive material exchange.1 This interface principally serves as a selective barrier for a range of exogenous materials, including ions, metabolites, growth factors, and toxins. As a plethora of recognition sites in the membrane translate biotic and abiotic environmental stimuli across the membrane, these are primary targets in studies toward a better understanding of signaling pathways and how biological responses are effected on the cellular level.2 Supported lipid bilayer (SLB) systems, typically formed on glass3 or PDMS,4 have proven to be a convenient platform for these studies, as the isolated lipid environment eliminates complexities and interferences from other cellular activities. These SLBs are easily tunable with a broad spectrum of compositional complexities ranging from single phospholipids to mixtures of lipids with embedded proteins and natural © XXXX American Chemical Society
Received: May 1, 2015 Accepted: July 20, 2015
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DOI: 10.1021/acsami.5b03809 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic diagram showing the process of vesicle deposition, desiccation, and devitrification upon hydration of the trehalose matrix on the modified SPR sensor chips. Each SPR chip is modified with ca. 10 nm of silica, applied by plasma-enhanced chemical vapor deposition to increase hydrophilicity and provide a fusogenic surface for the SUVs. The devitrification process releasing the SUVs takes place in the SPR flow cell environment.
hybrid bilayer membranes,10,22 where the hydrophobic tails of phospholipids adsorb to long-chain alkanethiol monolayers assembled on gold substrates. In previous work, we demonstrated that, by creating nanoscale layers of glass on gold surfaces,23 formation and characterization of stable bilayer membranes is also possible for SPR. Recently, a number of high-performance SPR imaging (SPRi) substrates have been developed that allow for ultrasensitive screenings of SLB systems in a high-throughput manner.24,25 These substrates featured thin coatings of silica, applied through advanced cleanroom techniques, attenuated background evanescent fields to yield higher dynamic response ranges, and allowed for the detection of proteinaceous toxins binding to receptorcontaining SLBs at low nanomolar concentrations. In this work, we report an approach that combines trehaloseassisted phospholipid vesicle deposition with SPRi for ondemand and label-free analysis of biomolecular interactions in an arrayed SLB system. Vesicle suspensions in trehalose were deposited on ultrathin (10 nm) layers of engineered glass deposited on gold substrates, desiccated, and directly used for analytical characterization once rehydrated (Figure 1). Lateral mobility properties of traditionally formed bilayers and those that stem from rehydrated lipids released from trehalose were compared on a variety of substrate surfaces, including Au/SiO2 glass coverslips and alkanethiol-modified Au. After empirically optimizing the flow rate conditions for the rehydration within the SPR flow cell, we studied the behavior of the generated lipid membranes by SPR in terms of the effective refractive index changes compared to traditionally formed membranes. Furthermore, affinity studies were carried out with cell membrane receptors, namely, gangliosides GM1, GM2, and GM3, in which the response signals for the binding of cholera
crucially depends on constant hydration of the fabricated array, as exposure to air results in a loss of structural integrity of the SLB. This translates to required on site fabrication of SLBs, with severe limits for even short-term storage and transport of SLB array substrates, facts that make commercialization and widespread adaption very challenging. However, a unique fabrication method was recently introduced that allows for the spatially defined deposition of matrix encapsulated lipid vesicles, followed by on-demand formation of solid-supported lipid bilayers once hydrated.16 With addition of a low molecular weight, nonreducing disaccharide, trehalose, to preformed vesicle suspensions, a strategy that mimics the natural preservation mechanisms encountered in drought-tolerant and anhydrobiotic organisms,17,18 vesicles remain intact during the vitrification of trehalose. Hydration leads to a devitrification of trehalose, removal of the sugar, and a concurrent release of the vesicles followed by their fusion into SLBs on fusogenic surfaces. Resulting lipid bilayers on glass were studied by fluorescence microscopy, and were shown having fluidity comparable to conventional SLBs, as well as being capable of maintaining embedded ligands and receptors in their active state throughout the trehalose vitrification and devitrification processes.16 Compared to fluorescence, label-free analytical methods such as surface plasmon resonance (SPR) allow for the characterization of molecular interactions in a highly efficient fashion without extra labeling or tagging steps, thereby eliminating potential interference and labor.19 SPR assays have successfully been applied toward studying a large variety of chemical and biological samples,20 and are user-friendly enough to be conducted in clinical settings.21 Many early studies utilizing SPR for investigating lipid membrane systems made use of B
DOI: 10.1021/acsami.5b03809 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
with acetone, leaving an elevated gold grid behind, defining the array elements on the SiO2. Prior to use, all SPR substrates were thoroughly rinsed alternately with DI water, isopropanol, and DI water and then dried in a stream of nitrogen. The hydrophilicity of SiO2 coated chips was additionally increased by exposure to an oxygen plasma for 60 s using a Harrick PDC-32G plasma cleaner (Harrick Plasma, Ithaca, NY). SPR and SPRi Instrumentation. A dual-channel SPR spectrometer, NanoSPR5-321 (NanoSPR, Chicago, IL), with a GaAs semiconductor laser light source (λ = 670 nm) was used for all spectroscopic SPR measurements. The device was equipped with a manufacturer-supplied high-refractive index prism (n = 1.61) and a 30 μL flow cell. Surface interactions at the gold interface were monitored using the resonance angle tracking mode. A detailed description of the SPR imaging instrumentation setup has been provided in previous work.26 In brief, each BK7 substrate coated with gold well arrays was mounted on an optical stage containing a 300 μL flow cell. Each array was put in contact with an equilateral SF2 prism (n = 1.616) using refractive index matching fluid (n = 1.616, Cargille Laboratories, Cedar Grove, NJ). The optical stage was fixed on a goniometer that allows manual selection of the incident light angle. A incoherent light source (LED, λ = 648 nm) was used for SPR excitation, and the reflected images were captured by a cooled 12bit CCD camera, Retiga 1300 (QImaging, Surrey, BC, Canada) with a resolution of 1.3 MP (1280 × 1024 pixels) and 6.7 μm × 6.7 μm pixel size. Injections of sample solutions into the flow cell were monitored in real time by recording changes in the reflectance every 300 ms inside the gold array wells and for reference purpose on the surroundings. Sensorgrams were obtained by averaging reflected light intensity over each array element using a home-built LabView program. Difference images were obtained by subtracting images collected with p-polarized from those recorded with s-polarized light. Desiccation of Vesicle Suspensions. An appropriate amount of preformed SUVs in 50 mM trehalose and 1× PBS (50 μL for SPR channels, 20 μL for fluorescence wells, 200 nL for SPRi array spots) was deposited on the chosen substrate surface and dried overnight in a vacuum desiccator; substrates were typically left under vacuum until use. In the case of the long-term storage assessment, substrates were moved from vacuum to ambient conditions after 12 h. Devitrification of Trehalose Coatings. Both the SPR and SPRi setups employ home-built fluidic systems at ambient temperature (∼23 °C), with 1× PBS used as the running buffer set to a flow rate of 6 mL/h unless otherwise noted. The substrates with trehalose suspended vesicles were placed directly into the SPR or SPRi instruments and rehydrated within the flow cell environment. Once a stable signal was obtained, indicating completion of the membrane formation and removal of excess material, the lipid bilayers were used for analytical studies. Fluorescence Microscopy and FRAP Analysis. Fluidity of membranes from traditional fusion of POPC vesicles and those from hydrated trehalose encapsulated POPC vesicles on different surfaces was examined using fluorescence recovery after photobleaching (FRAP). Supported lipid bilayers were formed on bare glass coverslips (Fisher Scientific, Pittsburgh, PA), glass coverslips covered with 10 nm of SiO2, and C18-modified glass coverslips. For the trehalose derived membranes, 20 μL of 2% (w/w) NBD-PC/98% (w/w) POPC in 50 mM trehalose and 1× PBS was deposited into 4.5 mm PDMS wells on top of the glass/modified Au substrates. Following an overnight dehydration in vacuum the vesicle suspension was rehydrated in 1× PBS buffer in situ the following day and rinsed thoroughly with DI water to remove unfused vesicles. For traditional membranes, 20 μL of 2% (w/w) NBD-PC/98% (w/w) POPC in 1× PBS was deposited into the PDMS wells and allowed to incubate for 1 h prior to rinsing with water. To assist with the identification of the focal plane for the bilayer under the microscope, a peripheral scratch on the membrane was made and used. Fluorescence microscopy was carried out on an inverted Leica TCS SP5 II (Leica Microsystems, Buffalo Point, IL) using the 488 nm Argon laser line and a 40× (NA 1.1) objective. Photobleaching at 1.5 mW for 500 ms and fluorescence recovery
toxin to differently prepared bilayers were found to be virtually identical. SPRi experiments showed no crosstalk between adjacent array elements. Individual binding responses of multiple monosialogangliosides across a multielement array were compared and exhibited excellent coherence, underscoring the utility of this versatile methodology for large-scale arrays. In addition to the fluid SLB arrays on Au/SiO2 substrates, we also show on-demand bilayer formation on hydrophobic surfaces resulting in hybrid bilayers and their characterization.
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EXPERIMENTAL SECTION
Materials and Reagents. Cholera toxin (CT) from Vibrio cholera, Triton X-100, 1-octadecanethiol (98%), and n-octadecyltrichlorosilane (OTS, 90+%) were from Sigma-Aldrich (St. Louis, MO). Trehalose was from Swanson Health Products (Fargo, ND). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1palmitoyl-2-{6-[(7-nitro-2−1,3-benzoxadiazol-4-yl)amino]hexanoyl}sn-glycero-3-phosphocholine (NBD-PC) were from Avanti Polar Lipids (Alabaster, AL). Monosialoganglioside GM1 (NH4+ salt) and monosialoganglioside GM2 (NH4+ salt) were from Matreya (Pleasant Gap, PA). Monosialoganglioside GM3 was from EMD Biosciences (La Jolla, CA). BK-7 glass substrates were from Corning (Painted Post, NY). Chromium and gold used for electron-beam evaporation were acquired as pellets of 99.99% purity from Kurt J. Lesker (Jefferson Hills, PA). Vesicle Preparation. An appropriate amount of lipid stock solution containing 95% (w/w) POPC and 5% (w/w) monosialoganglioside (GM1, GM2, or GM3) in chloroform was dried in a glass vial under nitrogen to form a thin lipid film. The vial containing lipids was then placed in a vacuum desiccator for at least 2 h to remove any residual solvent. The dried lipids were resuspended in 1× PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) to a lipid concentration of 2.0 mg/mL. After vigorous vortexing to remove all lipid remnants from the vial wall, the solution was probe sonicated for 20 min. The resuspended lipids were then centrifuged at 8000 rpm for 15 min to remove titanium particles from the sonicator probe tip. Thereafter, the supernatant was extruded through a polycarbonate filter (100 nm) to produce small, unilamellar vesicles (SUVs) of uniform size. If the vesicles were suspended in trehalose, the solution was diluted to a final concentration of 1.0 mg/mL PC in 50 mM trehalose using a trehalose/1× PBS mixture. If not, the solution was diluted to 1.0 mg/mL PC using 1× PBS. For fluorescence analysis, the vesicle preparation followed the same procedure with the addition of 2% (w/w) NBD-PC. All vesicle suspensions were applied within a week and stored at 4 °C before use. SPR Chip Fabrication. SPR and SPRi chips were fabricated using BK-7 glass microscope slides. First, BK-7 substrates were cleaned using boiling piranha solution (3:1 H2SO4/30% H2O2) for 30 min, followed by rinsing with DI water and drying under compressed air. For conventional SPR chips, 2.0 nm of chromium (0.5 Å/s) followed by 46.0 nm of gold (1.0 Å/s) was deposited using electron beam evaporation (Temescal, Berkeley, CA) at 5 × 10−6 Torr. To obtain a hydrophilic surface for lipid bilayer formation, 10 nm of SiO2 was deposited on top of the gold layer using plasma enhanced chemical vapor deposition (PECVD) with a Unaxis Plasmatherm 790 system (Santa Clara, CA). High-performance gold well SPRi chips were fabricated using previously developed methods24 (Figure S1, Supporting Information). A 2.0 nm layer of chromium and 51.0 nm of gold were deposited using electron beam evaporation on cleaned BK-7 glass substrates using the above protocol. The surface was then rendered hydrophilic with 10 nm coating of SiO2 deposited by PECVD. Subsequently, photoresist AZ5214E was spin coated on the gold/SiO2 at 4000 rpm, and the surface was patterned into mesas representing the final array spots using standard photolithography methods. After a second electron beam evaporation of 100.0 nm of gold, the photoresist was lifted off C
DOI: 10.1021/acsami.5b03809 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. FRAP analysis of supported lipid bilayers formed using direct, traditional vesicle fusion and trehalose assisted deposition methods on microscope coverslips and SiO2-modified SPR surfaces. Calculated values are the result of N = 3 experiments. (a) Fluorescence microscopy images showing bleaching and recovery of fluorescence due to redistribution of lipids over time. Scale bars represent 20 μm. (b) FRAP recovery curve of the devitrified membrane on modified SPR surface. (c) Diffusion coefficients. (d) Mobile fractions.
gold surface. As noted in previous work,23,30 the layer-by-layer assembly of polyelectrolytes and sodium silicate followed by high-temperature calcination is an effective and low-cost way to produce glassy silicate films of uniform, nanoscale thickness. Here we chose to explore membrane formation on SPR substrates on which silica (SiO2) is deposited by plasmaenhanced chemical vapor deposition (PECVD). This method offers the advantage of short process times (under 10 min) and remarkably smooth surfaces, which benefits lipid bilayer fluidity and minimizes optical scattering. Previous studies31,32 have established that these surfaces are characterized by low surface roughness values (rms
